PONE-D-24-48364Sleep phenotyping in a rat model of susceptibility to substance use disordersPLOS ONE

Dear Dr. de Vivo,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. In particular, your manuscript was evaluated by two expert reviewers, who found merit in your work, but also raised several issues. These issues included statistics and the interpretation of data obtained from alcohol-naive rats and were relevant to PLOS publication criteria n. 3 (experiments and statistics are performed to a high technical standard and described in sufficient detail) and n. 4 (conclusions are presented in an appropriate fashion and are supported by the data). I broadly agree with the Reviewers' comments, and encourage you to revise your manuscript accordingly.

Please submit your revised manuscript by Feb 12 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols .

We look forward to receiving your revised manuscript.

Kind regards and happy new year,

Alessandro Silvani, M.D., Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

1. When submitting your revision, we need you to address these additional requirements.

Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at 

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and 

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Thank you for stating in your Funding Statement: 

This study was supported by the Giovanni Armenise Harvard Foundation Career Development Award to LdV, and the National Institutes of Health grant AA017447 to MR and RC.

Please provide an amended statement that declares *all* the funding or sources of support (whether external or internal to your organization) received during this study, as detailed online in our guide for authors at http://journals.plos.org/plosone/s/submit-now.  Please also include the statement “There was no additional external funding received for this study.” in your updated Funding Statement. 

Please include your amended Funding Statement within your cover letter. We will change the online submission form on your behalf.

3. When completing the data availability statement of the submission form, you indicated that you will make your data available on acceptance. We strongly recommend all authors decide on a data sharing plan before acceptance, as the process can be lengthy and hold up publication timelines. Please note that, though access restrictions are acceptable now, your entire data will need to be made freely accessible if your manuscript is accepted for publication. This policy applies to all data except where public deposition would breach compliance with the protocol approved by your research ethics board. If you are unable to adhere to our open data policy, please kindly revise your statement to explain your reasoning and we will seek the editor's input on an exemption. Please be assured that, once you have provided your new statement, the assessment of your exemption will not hold up the peer review process.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: No

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1:  Major points:

1. Discussion could benefit from interpretation of the findings in relation to propensity for alcohol taking (e.g., how does potential effect on cognition, or learning and memory, impact alcohol drinking, or propensity to start drinking).

2. Given the msP rats are alcohol-naïve, some of the comparisons of results to sleep changes reported in individuals with AUD are not entirely applicable.

3. More information is expected to describe data acquisition software. Also, please make it clear whether data were individually hand scored or autoscored in batches. Autoscoring typically lacks reliability when compared to hand score.

4. Age of rats at time of recordings spans adolescence to adult? (average 58+/- 8 to 12 days). The range of age here is potentially problematic. Can data be separated and analyzed by age (although there may not a large enough group size for this and thus, additional data points for either age may be required). Sleep maturation during adolescence is an important factor to consider as sleep patterns undergo discernible changes including circadian rhythm shifts that could confound the early/late sleep measures here amongst other measures.

5. Why were sleep spindles only assessed during “early and late sleep,” or first and last 3 hours of the light period? what was happening in the other 6 hours? Why are these period of interest more than peak rest time?

Minor points for clarification:

1. If band pass on EEG was 0.1- 40 hz, why was beta power limited to 25 Hz?

2. For post hoc analyses, why fisher LSD in some cases and Sidak’s MCT in others?

3. Figure 1- “W” sometimes indicates Wistar and other times abbreviates “wake/waking,” please correct. For Figure 1A, figure says WAKE was n.s. but results text indicates significance- which is it? Statistical results are not detailed in Results for Figure 1C.

4. In results, the time is referred to as a percentage, but the graphs are showing time in minutes, please make them match.

5. Figure 4B, does not appear to be “change in uV2.”

Reviewer #2:  The manuscript by de Vivo et al. characterized the sleep phenotype of a preclinical rat model of patients with AUD. Overall this investigation was conducted adequately providing a baseline dataset for this specific rat model which, in future experiments, will be useful to deeper understanding the neural circuits at the basis of substance use vulnerability.

However, I have some major methodological and conceptual concerns:

- I believe this work would have been more complete if the authors had included groups exposed to alcohol (possibly using a longitudinal protocol). Limiting the sleep profiles of this model to baseline conditions is of course of interest, however, it would have been nice to see what specific sleep changes are due to alcohol exposure rather than genetic predisposition. Please comment on this.

- Similarly, the authors claim that the altered sleep profile in msP rats might entail memory consolidation and behavioral impairments. However, since they did not provide any data on these aspects, I suggest reporting these speculations in a specific limitation paragraph together with the reply of my previous point.

- I noticed the presence of outliers in several figures, and I feel that, at least some of them, importantly impacted on the statistics and the conclusion of the present work. In particular, fig. 1F (msp group), 1G (W-NREM, wistar group), 4C (msp group), 5B (w group). Did the authors check for the weight of them on their conclusions? Did they apply any automatic outlier detection?

- How did the authors correct for multiple comparisons in fig. 1c?

- Fig. 1D, the statistics in the text seems referring to a main ANOVA group effect rather than an interaction effect before applying the corrected post-hoc analysis.

I also have some minor concerns/suggestions:

- For figures 4E-4I, 5D-H, how did the authors choose to limit the SWA and spindle analyses to the first and last 3 hours of NREM sleep? Could you provide any reference for this choice?

- I would suggest including the sleep fragmentation index (n° of wakefulness episodes / total sleep time) to make easier (for the readers) the conclusions on this characteristic

- I would also suggest including representative hypnograms to readily appreciate the different sleep structure between groups.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy .

Reviewer #1: No

Reviewer #2: No

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org . Please note that Supporting Information files do not need this step.

PONE-D-24-48364R1Sleep phenotyping in a rat model of susceptibility to substance use disordersPLOS ONE

Dear Dr. de Vivo,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Specifically, I agree with Reviewer 1 that statistical issues were not adequately addressed in the first revision of your manuscript. Moreover, the Grubbs test is designed to identify single outliers from relatively normal distributions, which may have not been always the case in your dataset, and ANOVA is sensitive to non-normal distributions as well as to outliers. On the other hand, your results seem quite robust to different statistical approaches, and I understand your choice to provide a full representation of your results in the main manuscript. On these bases, I recommend that you perform a further minor revision of the analysis and presentation of your results to fully address these issues, possibly seeking the advice of a professional biostatistician. You may consider consistently using non-parametric tests instead of parametric tests to decrease the sensitivity of results to potential outliers. Please provide a detailed description of your statistical approach, including the identification and management of outliers, in the methods section of the main manuscript, and discuss any related limitation in the discussion section. Please submit your revised manuscript by May 21 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols .

We look forward to receiving your revised manuscript.

Kind regards,

Alessandro Silvani, M.D., Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1:  The authors have adequately addressed this reviewer's concerns, in large part, and the manuscript is much improved from the previous version. It is not clear, however, why the authors chose to present data both with (main figures) and without (supplemental figure S3) outliers. This reviewer would prefer the authors present only the figures and statistics that exclude outliers in the main body, and possibly only include figures with outliers as a supplement if they feel it necessary to show both. Reporting a statistically significant result that hinges on an outlier does little to enhance the reader's interpretation of the data.

Reviewer #2:  I have no furhter comments and I agree with the authors that the outlier analyses can be kept in the supplementary material.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy .

Reviewer #1: No

Reviewer #2: Yes:  Stefano Bastianini

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org . Please note that Supporting Information files do not need this step.

Sleep phenotyping in a rat model of susceptibility to substance use disorders

PONE-D-24-48364R2

Dear Dr. de Vivo,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager®  and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Alessandro Silvani, M.D., Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: